Since mixed chimerism has been shown in mouse and humanized mouse models (Project 3) to have the ability not only to induce central T cell tolerance, but also to prevent new antibody responses and turn off existing antibody responses, the establishment of xenogeneic mixed chimerism remains an attractive means for achieving xenograft tolerance. Before GalT-KO miniature swine were available, the failure of attempts to achieve mixed chimerism across the pig-to-primate barrier was attributed to high levels of primate natural antibodies to the Gal antigen. However, despite the absence of the Gal antigen on the cell surface of GalT-KO hematopoeitic cells (HSC), we have found that these cells are rapidly cleared from the circulation of baboons shortly after infusion, eluding the establishment of mixed chimerism. Our data indicate that two of the most important factors playing a role in this clearance are: 1) natural antibodies to non-Gal determinants, which are variably present in different baboons; and 2) absence of the species-specific cell-membrane protein, CD47, which is required to inhibit the innate immune system from rapidly clearing cells from the circulation. The goal of this proposal is to overcome these two barriers in order to establish mixed xenogeneic chimerism of GalT-KO HSC in baboons. Specifically, we will: 1) Optimize xenogeneic bone marrow engraftment by avoiding natural and induced anti-non-Gal antibodies; 2) Test effect of human CD47 on engraftment of porcine marrow in baboons using GalT-KO pig bone marrow transduced with a human CD47 expression vector; and 3) Test the effect of GalT-KO pig HSC engraftment in baboons, either alone or in combination with vascularized porcine thymus transplantation, on the induction of tolerance of GalT-KO renal xenografts. Aim 3 will apply the findings of Aims 1 and 2, as well as strategies developed in Project 1 of this Program Project, to the mixed chimerism approach for inducing transplantation tolerance to organ xenografts in this preclinical, discordant species combination. In addition, the experiments planned will provide basic information on xenogeneic stem cell engrafment and on the immunologic pathways responsible for xenogeneic rejection and tolerance induction in primates. As such, these studies should have both theoretical and practical implications for the eventual application of xenotransplantation as a clinical modality.